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• W2021320002 abstract "Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221–225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221–225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (K d(app)) = 44.1 ± 8.6 mm. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5–6-fold withK d(app) = 2.3 ± 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221–225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme. Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221–225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221–225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (K d(app)) = 44.1 ± 8.6 mm. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5–6-fold withK d(app) = 2.3 ± 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221–225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme. Protein C is a multi domain vitamin K-dependent plasma serine protease zymogen, which upon activation by the thrombin·TM 1The abbreviations TMthrombomodulinTM4–6thrombomodulin epidermal growth factor like domains 4–6GDPCGla domainless protein C from which the N-terminal residues 1–45 (in the chymotrypsin numbering system of Bode et al. (42Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar) were removed by the recombinant DNA methodsGDPC tryp/loopGDPC derivative in which residues Gly221-Tyr225 were replaced with corresponding residues of trypsinGDPC E80KGDPC derivative in which Glu80 was replaced with LysaGDPCactivated GDPCPPACKd-Phe-Pro-Arg chloromethyl ketonePEGpolyethylene glycolAPCactivated protein CPAGEpolyacrylamide gel electrophoresis complex, down-regulates the blood coagulation cascade by selectively inactivating factors Va and VIIIa (1Esmon C.T. Thromb. Haemostasis. 1993; 70: 1-5Crossref Scopus (51) Google Scholar, 2Walker F.J. Fay P.J. FASEB J. 1992; 6: 2561-2567Crossref PubMed Scopus (162) Google Scholar, 3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1636) Google Scholar). Monovalent and divalent cations regulate the protein C anticoagulant pathway at the level of both the protein C zymogen activation and the catalytic function of activated protein C (APC). In the zymogen activation process, binding of Ca2+ to the N-terminal Gla domain and the first epidermal growth factor like domain-1 is required for efficient assembly of the protein into the activation complexes on cell surfaces (4Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar, 5Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). In addition to the Gla domain, Ca2+ binding to a high affinity site in the catalytic domain induces a conformational change in the activation peptide of protein C that is required for rapid activation by the thrombin·TM complex (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). Following the activation, the non-catalytic domains of APC remain covalently linked to the serine protease domain to mediate the Ca2+- and Gla-dependent binding of APC to its specific cofactors, protein S or the endothelial cell protein C receptor (7Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (328) Google Scholar, 8Fukudome K. Esmon C.T. J. Biol. Chem. 1994; 269: 26486-26491Abstract Full Text PDF PubMed Google Scholar, 9Stearns-Kurosawa D.J. Kurosawa S. Mollica J.S. Ferrell G.L. Esmon C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10212-10216Crossref PubMed Scopus (457) Google Scholar). Although several previous studies have demonstrated that Ca2+ and other divalent cations stimulate the amidolytic and esterolytic activity of APC (10Amphlett G.W. Kisiel W. Castellino F.J. Biochemistry. 1981; 20: 2156-2161Crossref PubMed Scopus (61) Google Scholar, 11Hill K.A.W. Kroon M.E. Castellino F.J. J. Biol. Chem. 1987; 262: 9581-9586Abstract Full Text PDF PubMed Google Scholar), it is not known if occupancy of the Ca2+-binding site in the catalytic domain of APC is also required for its physiological function. thrombomodulin thrombomodulin epidermal growth factor like domains 4–6 Gla domainless protein C from which the N-terminal residues 1–45 (in the chymotrypsin numbering system of Bode et al. (42Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (823) Google Scholar) were removed by the recombinant DNA methods GDPC derivative in which residues Gly221-Tyr225 were replaced with corresponding residues of trypsin GDPC derivative in which Glu80 was replaced with Lys activated GDPC d-Phe-Pro-Arg chloromethyl ketone polyethylene glycol activated protein C polyacrylamide gel electrophoresis Similar to Ca2+, the monovalent cation Na+regulates the protein C anticoagulant pathway at the level of both zymogen activation and APC function. In this case, it is believed that Na+ allosterically regulates the activity of thrombin and that protein C is activated preferentially by the Na+-free form of thrombin, which is referred to as the slow form (12Dang Q.D. Vindigni A. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5977-5981Crossref PubMed Scopus (168) Google Scholar, 13Guinto E.R. Vindigni A. Ayala Y.M. Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11185-11189Crossref PubMed Scopus (47) Google Scholar). The Na+-bound or fast form of thrombin is believed to function in the procoagulant pathway by specifically activating fibrinogen (12Dang Q.D. Vindigni A. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5977-5981Crossref PubMed Scopus (168) Google Scholar,13Guinto E.R. Vindigni A. Ayala Y.M. Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11185-11189Crossref PubMed Scopus (47) Google Scholar). A dissociation constant of 30–100 mm for Na+ binding to thrombin has been reported (12Dang Q.D. Vindigni A. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5977-5981Crossref PubMed Scopus (168) Google Scholar, 14Wells C.M. Di Cera E. Biochemistry. 1992; 31: 11721-11730Crossref PubMed Scopus (226) Google Scholar). In contrast to protein C activation, however, it has been demonstrated that bovine APC displays a strict requirement for Na+ (or other monovalent cations) for expression of its amidolytic activity toward tri-peptide chromogenic substrates (15Steiner S.A. Castellino F.J. Biochemistry. 1998; 24: 609-617Crossref Scopus (22) Google Scholar). In previous studies an apparent dissociation constant of 87–129 mm for Na+ binding to bovine APC and activated Gla domainless protein C (aGDPC) has been reported (15Steiner S.A. Castellino F.J. Biochemistry. 1998; 24: 609-617Crossref Scopus (22) Google Scholar, 16Steiner S.A. Castellino F.J. Biochemistry. 1982; 21: 4609-4614Crossref PubMed Scopus (29) Google Scholar, 17Hill K.A.W. Castellino F.J. J. Biol. Chem. 1998; 261: 14991-14996Google Scholar). The exact role of Na+ in the activity of APC toward its physiological substrates, factors Va and VIIIa, is not known. In contrast to the divalent cation-binding sites of APC, the monovalent cation-binding site(s) of APC has not been identified. In the case of thrombin, a single metal-binding site for Na+ has been localized to a conserved loop consisting of residues 221–225 in the chymotrypsin numbering system (18Di Cera E. Guinto E.R. Vindigni A. Dang Q.D. Ayala Y.M. Wuyi M. Tulinsky A. J. Biol. Chem. 1995; 270: 22089-22092Crossref PubMed Scopus (226) Google Scholar, 19Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10653-10656Crossref PubMed Scopus (145) Google Scholar). The predicted consensus Na+-binding sequence in this loop is conserved in APC but not in trypsin (19Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10653-10656Crossref PubMed Scopus (145) Google Scholar). To determine whether this loop in APC binds Na+, we prepared a mutant lacking the Gla domain (GDPC) in which the 221–225 loop of GDPC (Gly221-Tyr225) was replaced with the corresponding residues of trypsin (Ala221-Pro225). Both the wild type and GDPC mutant (GDPC tryp/loop) were expressed in mammalian cells, and sufficient quantities of both derivatives were isolated for activation and characterization. In addition to this mutant, we recently prepared and characterized another GDPC mutant in which Glu80 in the Ca2+ binding loop of the molecule was replaced with Lys (E80K) (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). In this previous study, we demonstrated that the 70–80 loop in this mutant was stabilized in the Ca2+ conformer possibly by Lys80 forming a salt bridge with Glu70 (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). In the current study, the catalytic activities of the activated wild type, tryp/loop, and E80K GDPC (aGDPC) derivatives were monitored by their ability to hydrolyze several chromogenic substrates in both the absence and presence of Na+ and Ca2+. We found that unlike the wild type protease, the aGDPC tryp/loop mutant lost its ability to bind Na+ and no longer discriminated between various monovalent cations. The affinity of the mutant protease for Ca2+ was also impaired ∼16-fold. Interestingly, further study suggested that the affinity of the wild type aGDPC for Na+ in the presence Ca2+ was improved ∼20-fold, and the binding of aGDPC E80K to Na+ was of the high affinity type independent of Ca2+. These results suggest that the 221–225 loop is a Na+-binding site in APC, which is allosterically linked to the divalent cation-binding loop of the protease. The allosteric coupling of the two metal ion-binding loops in APC has likely played a role in the divergent evolution of this highly specific anticoagulant enzyme. The expression of Gla domainless protein C (GDPC) and GDPC E80K by the RSV-PL4 expression/purification vector system in human 293 cells has been previously described (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar, 20Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar). The 221–225 loop (Gly221-Tyr225) of GDPC was replaced with the corresponding sequence of trypsin (GDPC tryp/loop) by the polymerase chain reaction mutagenesis approach, and the mutant molecule was expressed in the same vector system as described previously (20Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar, 21Higuchi R. Krummel B. Saiki R. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2104) Google Scholar). Accuracy of the mutations was confirmed by sequencing prior to expression. The wild type and mutant zymogens were purified from the cell culture supernatants as described previously (20Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar). Human plasma protein C (22D'Angelo S.V. Comp P.C. Esmon C.T. D'Angelo A. J. Clin. Invest. 1986; 77: 416-425Crossref PubMed Scopus (127) Google Scholar), human plasma thrombin (23Owen W.G. Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 594-605Abstract Full Text PDF PubMed Google Scholar), human factor Va (24Smirnov M.D. Safa O. Regan L. Mather T. Stearns-Kurosawa D.J. Kurosawa S. Rezaie A.R. Esmon N.L. Esmon C.T. J. Biol. Chem. 1998; 273: 9031-9040Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), bovine antithrombin (25Owen W.G. Biochim. Biophys. Acta. 1975; 405: 380-387Crossref PubMed Scopus (110) Google Scholar), recombinant human prethrombin-1 (26Rezaie A.R. Biochemistry. 1996; 35: 1918-1924Crossref PubMed Scopus (39) Google Scholar), and recombinant human thrombomodulin fragment 4–6 (TM4–6, the minimum fragment of TM required as a cofactor for thrombin activation of protein C) (20Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar) were prepared by the cited methods. The chromogenic substrate Spectrozyme PCa (SpPCa) was purchased from American Diagnostica (Greenwich, CT), and S2266 and S2238 were purchased from Kabi Pharmacia/Chromogenix (Franklin, OH). The initial rate of protein C activation by the thrombin·TM4–6 complex (1 nm thrombin in complex with 100 nm TM) was measured as a function of different concentrations of GDPC or GDPC tryp/loop in 0.1 m NaCl, 0.02 m Tris-HCl, pH 7.5 (TBS), containing 0.1% polyethylene glycol 8000 (PEG 8000), and 2.5 mmCa2+ at room temperature. After inhibition of thrombin activity by antithrombin, the initial rates of activation were measured from the rate of activated protein C generation in an amidolytic activity assay using 1 mm SpPCa in TBS buffer containing 0.1% PEG 8000 and 2.5 mm Ca2+. The rate of hydrolysis was measured at 405 nm at room temperature in aV max kinetic plate reader (Molecular Devices, Menlo Park, CA). Fixed reaction times of 5 min were employed for each GDPC concentration. Over this time only the initial rate of activation was measured and less than 1% substrate was activated. The concentration of active protein C derivatives in reaction mixtures was determined by reference to a standard curve, which was prepared by total activation of GDPC at the time of each experiment. This was accomplished by activation of 1 μm each protein C derivative with 10 nm thrombin in complex with 100 nm TM4–6 and 2.5 mm Ca2+ for 90 min at 37 °C. Under these experimental conditions, all protein C zymogens were completely activated in less than 30 min. TheK m and k cat values of protein C activation were calculated from the Michaelis-Menten equation. Protein C activation was also carried out on thrombin·TM complex assembled on Affi-Gel 10 (Bio-Rad) as described previously (22D'Angelo S.V. Comp P.C. Esmon C.T. D'Angelo A. J. Clin. Invest. 1986; 77: 416-425Crossref PubMed Scopus (127) Google Scholar). The active site concentrations of the aGDPC derivatives were determined by an active site-specific immunoassay using BioCap-FPR-ck (biotinyl-ε-aminocaproyl-d-phenylalanine prolylarginine chloromethyl ketone) (Hematologic Technologies Inc., Essex Junction, VT) as described previously (27Rezaie A.R. J. Biol. Chem. 1996; 271: 23807-23814Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The aGDPC derivatives were desalted by passing through PD-10 (Amersham Pharmacia Biotech) gel filtration columns equilibrated with Chelex-treated (Bio-Rad) 5 mm Tris-HCl, pH 8.0, buffer containing 0.1% PEG 8000 and stored at −80 °C. The steady-state kinetic parameters of wild type and mutant enzyme toward S2266 (d-Val-Leu-Arg-p-nitroanilide) was performed at room temperature in 5 mm Tris-HCl, pH 8.0, containing 0.1% PEG 8000, 2.5 mm CaCl2, and 0.2 m of different monovalent chloride salts (LiCl, NaCl, KCl, and choline chloride). The rate of hydrolysis was measured at 405 nm at room temperature in a V max kinetic plate reader as described above. The apparent K m andk cat values for substrate hydrolysis were calculated from the Michaelis-Menten equation. The specificity constant for each chloride salt was expressed as the ratio ofk cat/K m. The specificity constant for the bulky monovalent cation choline (Ch+) was used as a reference to determine the monovalent cation specificity as described by Dang and Di Cera (19Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10653-10656Crossref PubMed Scopus (145) Google Scholar). The concentration of S2266 ranged from 20 μm to 15 mm depending on theK m values, and the concentration of enzymes ranged from 10 to 100 nm depending on thek cat values. The values forK d(app) of Na+ binding to each protease was determined from the effect of varying concentrations of Na+ on the activity of the protease toward three synthetic substrates SpPCa, S2266, and S2238 in both the absence (Chelex-treated buffer) and presence of 2.5 mmCa2+. In all experiments a constant ionic strength of 0.2m was maintained by addition of choline chloride. This procedure has been commonly used in the past to study the effect of monovalent cations on the catalytic function of various enzymes (12Dang Q.D. Vindigni A. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5977-5981Crossref PubMed Scopus (168) Google Scholar,19Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10653-10656Crossref PubMed Scopus (145) Google Scholar). The values for K d(app) were calculated from the hyperbolic increase in the rate of substrate hydrolysis as a function of increasing Na+concentrations. The time course of human factor Va inactivation by activated GDPC was measured by a two-stage assay. In the first stage, factor Va (50 nm) was incubated with aGDPC (5 nm) at room temperature in 0.02 m Tris-HCl, pH 7.5, containing 0.15 m NaCl or 0.15 m KCl and 0.1% PEG 8000. This stage of the assay was carried out in both the absence and presence of 2.5 mm Ca2+. For this purpose, aGDPC and factor Va (in 5 mm Ca2+) were passed through two separate PD-10 gel filtration columns equilibrated with Chelex-treated 0.02 m Tris-HCl, 0.1% PEG 8000 and used immediately in kinetic experiments. In the second stage, at different time intervals (0–12.5 min) the remaining activity of factor Va was determined by measuring its ability to accelerate factor Xa activation of recombinant human prethrombin-1 as described previously (28Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar). The apparent K m andk cat values for substrate hydrolysis were calculated from the Michaelis-Menten equation, and the affinity of Na+ for each aGDPC derivative (K d(app)) was determined by nonlinear regression fits of data to a rectangular hyperbola using ENZFITTER (R. J. Leatherbarrow, Elsevier, Biosoft). All values are the average of at least 3–5 independent measurements ±S.D. Recombinant wild type and mutant GDPC derivatives were expressed in 293 cells and isolated as described under “Experimental Procedures”. SDS-PAGE analysis (Fig. 1) indicated that both derivatives expressed as two subforms with identical apparent molecular weights that correspond to α and β protein C that are glycosylation variants observed previously with this protein (20Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar). Under reducing conditions, a light chain was also observed with both derivatives. With both proteins a fraction of the protein samples remained non-reducible suggesting that the GDPC derivatives were expressed as a mixture of single and two-chain proteins. This property has been also observed for both recombinant and plasma-derived human protein C in the past (29Miletich J.P. Broze Jr., G.J. J. Biol. Chem. 1990; 265: 11397-11404Abstract Full Text PDF PubMed Google Scholar, 30Grinnell B.W. Walls J.D. Gerlitz B. Berg D.T. McClure D.B. Ehrlich H. Bang N.U. Yan S.B. Adv. Appl. Biotechnol. Ser. 1991; 11: 29-63Google Scholar). We have previously demonstrated that both single and double-chain APC derivatives have identical catalytic activities (31Rezaie A.R. Esmon C.T. Biochemistry. 1995; 34: 12221-12226Crossref PubMed Scopus (28) Google Scholar). These results suggest that the mutation does not alter the post-translational modifications or the processing of the protein. With the mutant GDPC, however, a minor band migrating at ∼35 kDa was also observed (Fig. 1). The nature of this band was not characterized. Comparison of the initial rate of protein C activation by the thrombin·TM4–6 complex as a function of different zymogen concentrations suggested that both GDPC derivatives were activated at a similar rate (Fig. 2). SDS-PAGE analysis of the activated products indicated that both zymogens were completely converted to activated forms (data not shown). This was consistent with the observation that the concentrations of enzymes as determined by the active site titration were similar (within 80%) with the values calculated based on the absorbance at 280 nm. To determine whether the mutant aGDPC can bind Na+, the effect of increasing concentration of Na+ on the activity of aGDPC derivatives toward the chromogenic substrates S2266, S2238, and SpPCa was studied. Since the catalytic domain of APC contains a Ca2+-binding site (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar), these studies were carried out both in the absence and presence of Ca2+. In the absence of Ca2+, the amidolytic activity of aGDPC was strongly dependent on the presence of Na+ in the reaction buffer (Fig. 3 A). The amidolytic activity of aGDPC was enhanced with increasing concentrations of Na+ and reached saturation with aK d(app) of 44.1 ± 8.6 mm. In a previous study, the interaction of Na+with bovine aGDPC was shown to be cooperative with a Hill coefficient of 1.5 (17Hill K.A.W. Castellino F.J. J. Biol. Chem. 1998; 261: 14991-14996Google Scholar). A similar cooperativity for Na+ binding to human aGDPC was also observed in this study in the absence of Ca2+ (data not shown). However, when the ionic strength of medium was adjusted to 0.2 m with choline chloride, no significant cooperativity was observed, and the nonlinear regression fits of data to a rectangular hyperbola was found to be suitable for obtaining the K d(app) values (Fig.3 A). In the presence of Ca2+, no cooperativity for Na+ binding to aGDPC was observed irrespective of whether the ionic strength of medium was adjusted to 0.2 mwith choline chloride. In this case an ∼5–6-fold stimulation of the amidolytic activity of aGDPC was observed at saturating Na+concentrations (Fig. 3 A). Interestingly, the affinity of Na+ for the protease was also improved ∼20-fold in the presence of Ca2+ (K d(app) = 2.3 ± 0.3 mm), and at higher concentrations of Na+ (>60 mm) the activity of aGDPC was slightly diminished. A similar improvement in the affinity of Na+ for the protease in the presence of Ca2+was observed at physiological temperature, although theK d(app) values were slightly elevated (141.0 ± 20.9 and 5.3 ± 0.3 mm in the absence and presence of 2.5 mm Ca2+, respectively). To ensure that this effect of Ca2+ on the Na+binding properties of APC was not a phenomenon related to the Gla domainless form of APC or an effect related to a particular substrate, the amidolytic activity of plasma-derived APC was monitored as a function of Na+ in both the absence and presence of Ca2+ with three different chromogenic substrates (S2266, S2238, and SpPCa). In all cases, similar results were obtained (data not shown). Only the results with SpPCa hydrolysis for aGDPC at room temperature are presented in these figures. It is worth noting that in previous studies, the amidolytic activity of bovine APC displayed a strict requirement for Na+ (16Steiner S.A. Castellino F.J. Biochemistry. 1982; 21: 4609-4614Crossref PubMed Scopus (29) Google Scholar). In the current study, we noticed some base-line amidolytic activity for human aGDPC in the absence of Na+ and Ca2+(Fig. 3 A). However, we believe that our results are consistent with the literature since a similar base-line activity was also observed in previous studies (15Steiner S.A. Castellino F.J. Biochemistry. 1998; 24: 609-617Crossref Scopus (22) Google Scholar, 16Steiner S.A. Castellino F.J. Biochemistry. 1982; 21: 4609-4614Crossref PubMed Scopus (29) Google Scholar). It was previously suggested that the base-line amidolytic activity of APC in the absence of Na+ and Ca2+ may be due to the presence of other monovalent cations such as Tris+ and/or choline (Ch+) in the reaction buffer (15Steiner S.A. Castellino F.J. Biochemistry. 1998; 24: 609-617Crossref Scopus (22) Google Scholar, 16Steiner S.A. Castellino F.J. Biochemistry. 1982; 21: 4609-4614Crossref PubMed Scopus (29) Google Scholar). The Na+ concentration dependence of the amidolytic activity of aGDPC tryp/loop mutant was studied in a similar fashion. In the absence of Ca2+, no K d(app)could be estimated for the Na+ interaction with the mutant since no saturation of Na+ binding to the mutant was observed up to 400 mm (data are presented for up to 200 mm NaCl in Fig. 3 B). No attempt was made to increase the concentration of Na+ above 400 mmsince the effect of high ionic strength on the structure of the enzyme is not known. However, in the presence of Ca2+, the amidolytic activity of the mutant was insensitive to the absence or presence of Na+ (Fig. 3 B). These results clearly suggest that the mutant has lost its ability to bind Na+and further suggest that Ca2+ binding to aGDPC has a profound effect on the ability of the protease to interact with Na+. There are two known Ca2+-binding sites on aGDPC that can influence the Na+ binding properties of the protease. The first Ca2+-binding site resides in the epidermal growth factor like domain-1 of the light chain and the other was localized to the C-terminal catalytic domain of APC (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar, 32Öhlin A. Landes G. Bourdon P. Oppenheimer C. Wydro R. Stenflo J. J. Biol. Chem. 1988; 263: 19240-19248Abstract Full Text PDF PubMed Google Scholar). The Ca2+-binding site in the catalytic domain is located on a loop between residues Glu70 and Glu80, analogous to the Ca2+ binding loop in trypsin (33Bode W. Schwager P. J. Mol. Biol. 1975; 98: 693-717Crossref PubMed Scopus (411) Google Scholar). Previously, we prepared and characterized a GDPC derivative in which Glu80 was replaced with Lys (E80K) (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). In that study, we demonstrated that the catalytic domain of the mutant was stabilized in the Ca2+ conformer possibly as a result of Lys80 in the mutant forming a salt bridge with Glu70 (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). To characterize further the Na+-binding site of APC and determine the Ca2+-binding site responsible for altering the Na+ binding properties of the protease, the amidolytic activity of aGDPC E80K toward SpPCa was studied as a function of different concentrations of Na+. Interestingly, theK d(app) values for Na+binding to this mutant was of high affinity and insensitive to the absence or presence of Ca2+ (5.1 ± 0.7 mmin the absence of Ca2+ and 5.0 ± 0.6 mmin the presence of Ca2+) (Fig.4). TheK d(app) of Na+ for the mutant did not change even if the amidolytic activity of the mutant was monitored in the presence of 0.1 mm EDTA or EGTA to chelate divalent metal ions. It is known that there is an ∼20% Ca2+ stimulation of the amidolytic activity of aGDPC toward chromogenic substrates with aK d(app) of ∼50 μm (6Rezaie A.R. Mather T. Sussman F. Esmon C.T. J. Biol. Chem. 1994; 269: 3151-3154Abstract Full Text PDF PubMed Google Scholar). To determine whether mutagenesis of the 221–225 loop influences the affinity of the 70–80 loop for binding to Ca2+, the amidolytic activity of aGDPC tryp/loop was monitored as a function of increasing Ca2+ concentrations. Two interesting observations emerged. First, unlike the small effect of Ca2+ on the amidolytic activity of aGDPC, Ca2+stimulated the amidolytic activity of the mutant toward the chromogenic substrates 8–10-fold (data not shown). Second, theK d(app) for Ca2+ binding to the aGDPC tryp/loop mutant increased to ∼800 μm, representing about 16-fold weaker interaction than that observed for Ca2+ binding to aGDPC. Taken together, these results suggest that occupancy of one metal-binding site of aGDPC with its specific metal cation influences the affinity of the other site for its specific metal ligand. We conclude that the 70–80 and 221–225 loops of aGDPC are allosterically linked. It is known that other monovalent alkali cations can substitute for Na+ in stimulation of the amidolytic activity of bovine APC and that the activity increases in parallel with increasing cation radius (16Steiner S.A. Castellino F.J. Biochemistry. 1982; 21: 4609-4614Crossref PubMed Scop" @default.
• W2021320002 created "2016-06-24" @default.
• W2021320002 creator A5019234325 @default.
• W2021320002 creator A5078346797 @default.
• W2021320002 date "1999-02-01" @default.
• W2021320002 modified "2023-10-10" @default.
• W2021320002 title "Identification and Characterization of the Sodium-binding Site of Activated Protein C" @default.
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